The membrane protein gene superfamily of G-protein coupled receptors (GPRs) has been characterized as having seven putative transmembrane domains. The domains are believed to represent transmembrane .alpha.-helices connected by extracellular or cytoplasmic loops. Of the 74 sequenced members of this G-protein receptor superfamily, the shortest sequence of 324 amino acids represents the rat mas oncogene and the longest, of 744 amino acids, represents the human thyroid-stimulating hormone (TSH) receptor. GPRs thus include a wide range of biologically active receptors, such as hormone-, viral-, growth factor- and neuroreceptors.
G-protein coupled receptors have been characterized as including these seven conserved hydrophobic stretches of about 20-30 amino acids, connecting at least 8 divergent hydrophilic loops. The G-protein family of coupled receptors includes dopamine receptors which bind in a noncovalent but high affinity manner to neuroleptic drugs used for treating psychotic and neurological disorders. For example, the dopamine D.sub.2 receptor includes these transmembrane domains, two of which (TM III and TM V; see below) have been implicated by site-selective mutagenesis to demonstrate functional, association with D.sub.2 ligands.
Transmembrane domains of G-protein coupled receptors are designated TM1, TM2, TM3, TM4, TMS, TM6 and TM7. TM4, TM5, TM6 and TM7 are the most highly conserved and are postulated to provide sequences which impart biological activity to GPRs. Most GPRs have single conserved cysteine residues in each of the first two extracellular loops which form disulfide bonds that are believed to stabilize functional protein structure. TM3 is also implicated in signal transduction.
Phosphorylation and lipidation (palmitylation or farnesylation) of cysteine residues can influence signal transduction of some GPRs. Most GPRs contain potential phosphorylation sites (e.g., serine or theronine residues) within the third cytoplasmic loop and/or the carboxy terminus. For several GPRs, such as the .beta.-adrenoreceptor, phosphorylation by protein kinase A and/or specific receptor kinases mediates receptor desensitization.
Non-limiting examples of GPRs include cAMP receptors, adenosine receptors, .beta.-adrenergic receptors, muscarinic acetylcholine receptors, .alpha.-adrenergic receptors, serotonin receptors (5-HT), histamine H2 receptors, thrombin receptors, kinin receptors, follicle stimulating hormone receptors, opsins and rhodopsins, odorant receptors, cytomegalovirus receptor, etc. See e.g., Probst et al DNA and Cell Biology 11:1-20(1992), which is entirely incorporated herein by reference.
The ligand binding sites of GPRs are believed to comprise a hydrophilic socket formed by several GPR transmembrane domains, which socket is surrounded by hydrophobic residues of the GPRs. The hydrophilic side of each GPR transmembrane helix is postulated to face inward and form the polar ligand binding site. TM3 has been implicated in several GPRs as having a ligand binding site, such as including the TM3 aspartate residue. Additionally, TM5 serines, a TM6 asparagine and TM6 or TM7 phenylalanines or tyrosines are also implicated in ligand binding.
GPRs can be intracellularly coupled by heterotrimeric G-proteins to various intracellular enzymes, ion channels and transporters. See, e.g., Johnson et al Endoc. Rev. 10:317-331(1989) ; and Birnbaumer et al Blochem. Biophys. Acta 1031:163-224(1990) which references are incorporated entirely herein by reference. GPR agonist binding catalyzes the exchange of GTP for GDP on the .alpha.-subunit of the G-protein. Different G-protein .alpha.-subunits preferentially stimulate particular effectors to modulate various biological functions in a cell. Phosphorylation of cytoplasmic residues of GPRs has been identified as an important mechanism for the regulation of G-protein coupling of some GPRs.
As a non-limiting example of a GPR ligand, dopamine (3,4-dihydroxyphenethylamine) is a critical neurotransmitter in the central nervous system (e.g., in the substantial nigra, midbrain, and hypothalamus). Since the elucidation of the ascending mesolimbic and nigrostriatal pathways, these pathways have been found to be critical in the control of both motor initiation (nigrostriatal) behavior and affectire (mesolimbic) behavior. The clinical efficacy of the major neuroleptic antipsychotic medications has been found to correlate with the respective affinities of these agents for the dopamine D.sub.2 receptor in the brain. A dopaminergic role in the symptomatology of the major psychoses has thus been hypothesized, although it is unclear if dopamine alone is etiological, (see, e.g., Davis et al. Am. J. Psych. 148:1474-1476 (1991)). Nonetheless, this hypothesis has served as a stimulus for current research in this area.
One model for studying possible interactions of G-protein coupled receptors with their ligands has emerged from site-directed mutagenesis and biochemical analysis of the .beta.-adrenergic receptor, as well as from biophysical analysis of the interaction of retinal with opsin.
According to such a model, the binding of a GPR ligand to a G-protein coupled receptor involves multiple interactions between functional groups on the GPR ligand and residues within the hydrophophilic binding site of the receptor.
While a number of the amino acid residues in the dopamine D.sub.2 receptor have been postulated to participate in D.sub.2 ligand binding, based on results obtained from site-directed mutagenesis studies and photoaffinity labeling studies performed on the .beta.-adrenergic receptor, such studies have failed to specifically determine which residues are actually involved in binding in the D.sub.2 system. Sibley et al. Soc. Neurosci. Abs. 17:36.10, 324.5, 324.6 (1991).
The clinical use of neuroleptics has provided a means for treating patients suffering from psychotic disorders. Short-term use of neuroleptics is indicated in several types of psychotic disorders, e.g., acute psychotic episodes, regardless of type; exacerbations of schizophrenia; acute manic excitement while deferring use of lithium or awaiting onset of its effects; adjunctive therapy for major depression with prominent psychotic symptoms, or when an antidepressant or ECT alone is not successful; for agitation in delirium, dementia, or severe mental retardation while seeking to identify and treat the primary basis of the problem; in certain chronic, degenerative, or idiopathic neuropsychiatric disorders with dyskinesias, such as Huntington's disease or Gilles de la Tourette's syndrome; or for ballism or hemiballism; childhood psychoses or apparently allied conditions marked by severe agitation or aggressive behavior; miscellaneous medical indications, notably nausea and vomiting, or intractable hiccups.
Additionally, continuous long-term use of neuroleptics is indicated in many psychotic disorders, such as (for more than six months) (i) primary indications such as Schizophrenia, Paranoia.sup.a,b, Childhood psychoses, some degenerative or idiopathic neuropsychiatric disorders (notably, Huntington's disease and Gilles de la Tourette's syndrome); (ii) secondary indications such as extremely unstable manic-depressive or other episodic psychoses (unusual), otherwise unmanageable behavior symptoms in dementia, amentia, or other brain syndromes; and (iii) questionable indications such as chronic characterological disorders with schizoid, "borderline," or neurotic characteristics; substance abuse; or antisocial behavior, recurrent mood disorders. See, e.g., Baldessarini, Chemotherapy in Psychiatry, Revised and Enlarged Edition, Harvard University Press, Cambridge, Mass., (1985), the contents of which is entirely incorporated herein by reference.
Neuroleptics are also referred to as neuroplegics, psychoplegics, psycholeptics, antipsychotics and major tranquilizers, but are sometimes distinguished from non-neuroleptic anti-psychotics. Neuroleptics have recently been characterized as an agent that produces sedative or tranquilizing effects, and which also produces motor side effects, such as catalepsy or extrapyramidal symptomatology. Nonlimiting representative examples of neuroleptics include phenothiazine derivatives (e.g., chlorpromazine); thioxanthine derivatives (e.g., thiothixene); butyrophenone derivatives e.g., haloperidol); dihydroindolone (e.g., molindone); dibenzoxazepine derivatives (e.g., loxapine); and "atypical" neuroleptics (e.g., sulpiride, remoxipiride pimozide and clozapine). See Berstein Clinical Pharmacology Littleton, Mass. :PSG Publishing (1978); Usdin et al Clinical Pharmacology in Psychiatry New York:Elsevier North-Holland (1981); and Baldessarini, supra, (1985); and , which references are herein entirely incorporated by reference.
The term "atypical neuroleptics" has been used to describe antipsychotic neuroleptics that produce few or no extrapyramidal side effects and which do not cause catalepsy in animals (See, e.g., Picket et al, Arch. Gen. Psychiatry 49:345 (May 1992). Alternatively, atypical neuroleptics, such as clozapine, have been described as those neuroleptics which have a higher affinity for D.sub.4 and D.sub.1 sites than for D.sub.2 sites (See, e.g., Davis et al Amer. J. Psych. 148:1474, 1476 (November 1991).
The long term use of all known anti-psychotics, such as neuroleptics or non-neuroleptic antipsychotics, has resulted in serious side effects, as present in Table I, such as persistent and poorly reversible motoric dysfunctions (e.g., tardive dyskinesia) in a significant number of patients. These side effects are especially prevalent in geriatric populations, and adequate pharmacological treatment of these debilitating morotic dysfunctions is not currently available. This problem has severely limited the long-term, clinical administration of these agents.
TABLE I __________________________________________________________________________ Neurological Side Effects of Neuroleptic-Antipsychotic Drugs Period of Reaction Features maximum risk Proposed mechanism Treatment __________________________________________________________________________ Acute dystonia Spasm of muscles of 1-5 days Dopamine excess? Antiparkinsonism agents are tongue, face, neck, Acetylcholine excess? diagnostic and curative back; may mimic (i.m. or i.v., then p.o.) seizures; not hysterical Parkinsonism Bradykinesia, rigidity, 5-30 days Dopamine blockade Antiparkinsonism agents variable tremor, mask- (rarely (p.o); dopamine agonists facies, shuffling gait persists) risky? Akathisia Motor restlessness; 5-60 days Unknown Reduce dose or change drug patient may experience (commonly low doses of propranolol; anxiety or agitation persists) antiparkinsonism agents or or benzodiazepines may help Tardive Oral-facial dyskinesia; 6-24 months Dopamine excess? Prevention best; treatment dyskinesia choreo-athetosis, some- (worse on unsatisfactory; slow spontaneous times irreversible, withdrawal) remission rarely progressive "Rabbit" Perioral tremor (late Months or Unknown Antiparkinsonism agents; reduce syndrome parkinsonism variant?); years dose of neuroleptic usually reversible Malignant Catatonia, stupor, Weeks Unknown Stop neuroleptic; antiparkinsonism syndrome fever, unstable pulse agents usually fail; bromocriptine and blood pressure; often helps; dantrolene variable; myoglobinemia; can general supportive care crucial be fatal __________________________________________________________________________ .sup.a There may be an increased risk of hypotension on interacting high doses of propranolol with some antipsychotic agents; clonidine may also b effective at doses of 0.2-0.8 mg/day, but carries a high risk of hypotension (Zubenko et al., Psychiatry Res. 11: 143, 1984).
In addition, clozapine, although apparently capable of producing less motor side effects, can cause irreversible, potentially fatal agranulocytosis in a minority of patients administered the drug. Such serious side effects limit the use of clozapine to patients who are resistant to treatment with other neuroleptics.
Antipsychotics have a variety of significant pharmacological effects, e.g., as presented in the following Tables II and III.
TABLE II __________________________________________________________________________ Comparative Pharmacology of Neuroleptics Phenothiazine Thioxanthene Butyrophenone Alkaloid Derivative Derivative Derivative Pharmacologic Actions Chlorpromazine Thiothixene Haloperidol __________________________________________________________________________ Antipsychotic Yes++ Yes++ Yes++++ Antiemetic Yes+++ Not tested Yes+++ Hypothermia Yes+ Yes+ No Hypotension Yes++ Yes+++ + Parkinsonism Yes++ Yes+ Yes++++ Antiadrenergic Yes++ Yes+++ + Anticholinergic Yes+ Yes+ Negligible Antihistaminic Yes+ Negligible Negligible Releases NE, DA No No No Blocks DA Yes++ Yes+ Yes++++ Blocks NE Yes++ Yes+++ Yes+ Central sympathetic Yes++ Yes+ Yes+++ suppressant __________________________________________________________________________ Chlorpromazine, thiothixene, and haloperidol decrease the functional availability of dopamine (DA) and norepinephrine (NE) by blocking the dopamine receptor sites in the basal ganglia and norepinephrine receptor sites in thalamic and hypothalamic areas. Reserpine simply reduces the concentrations of norepinephrine and dopamine in these areas. Both of these actions result in suppression of central sympathetic activity. + .fwdarw. ++++ indicate, from very weak to very strong effects.
TABLE III ______________________________________ Comparative Pharmacology of Antipsychotics Extrapyramidal Adrenergic Drug Sedation Blockage Reaction ______________________________________ Chlorpromazine High Moderate to high Moderate Chlor- High High Low to moderate prothixene Haloperidol Low Low High Molindone Moderate Moderate Moderate to high Loxapine High Low to moderate High ______________________________________
See Ebadi, PHARMACOLOGY, Little, Brown and Co., Boston, 61-65 (1985); Cattabeni et al Adv. Biochem. Psychopharmacology 24:275 (1980). Baldessarini, supra, which references are herein incorporated entirely by reference.
However, despite the face that thousands of neurolepticor antipsychotic-type compounds have been synthesized and reported in the literature, such compounds which lack serious side effects and which have sufficient pharmacological activity, have not been disclosed.
Alternative to dopamine receptor GPRs, as presented above, other neuroreceptor GPRs are involved in neurological pathologies, and drugs such as neuroreceptor GPR binding agents, presently used for treating these pathologies, also suffer from similar side effects as those of neuroleptics, as presented above.
Other GPRs are also involved in receptor-related pathologies, such as hormone related GPRs involved in endocrine related pathologies,
Accordingly, there is a need to provide G-protein coupled receptor binding agents, including neuroreceptor and endocrine receptor GPRs, which do not produce such deleterious and debilitating side effects as those produced by known agents, such as neuroleptics, which can be used for therapy or diagnosis of GPR related pathologies.
Citation of documents herein is not intended as an admission that any of the documents cited herein is pertinent prior art, or an admission that the cited documents are considered material to the patentabilty of the claims of the present application. All statements as to the date or representations as to the contents of these documents are based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.